The present invention relates to a method for the stimulation of pancreatic cells to produce and/or excrete insulin, the stimulation being provided in connection with culture, transport, storage, isolation, transplantation and similar. The pancreatic cells considered herein include mature insulin-producing xcex2-cells and their precursors, fetal xcex2-cells.
The present invention also relates to a method for in-vitro cultivation of human adult and/or fetal cells, in particular cell types considered difficult or impossible to cultivate in existing media, such as stem cells, fibroblasts, neural cells and epithelial cells.
The present invention also relates to a method for in-vitro cultivation of human adult and fetal cells, a method for the expression of peptides in bacteria, in particular E. coli, a method for wound healing, a method for skin treatment, and a method for preservation and/or transport and/or storage of tissue and organ transplants.
Furthermore the present invention relates to a means for carrying out said methods and to uses of the means.
Transplantation of insulin-producing pancreatic cells is an attractive alternative to the presently used life-long administration of insulin to diabetics whose pancreas does not produce this vital hormone or only does produces it in insufficient amounts. At present pancreatic transplantation means transplantation of the entire organ or a major part thereof. The transplantation of pancreatic islets only, that is, cell clusters comprising xcex2-cells, is considered to be in an early experimental state. Transplantation of pancreatic islets only should be more attractive than transplantation of the entire pancreas since the availability of such organs of human origin is limited. The preparation of viable porcine or human islets for clinical investigation is known (Gray D W R et al., Diabetes 1984, 33:1055; Ricordi C et al., Diabetes 1990, 37:1377) and such implantation has provided encouraging results (for instance, Scharp D W et al., Diabetes 1990, 39:515). The remaining problem, however, is how to provide sufficient amounts of these cell preparations, and how to stimulate them to excrete substantial amounts of insulin. For various reasons it has been proposed to use fetal pancreatic tissue (for a survey, se: Andersson A and Sandler S., Transplantation Reviews 6 (1992) 20-38; reference to this survey also comprises reference to research papers cited therein).
It is believed (cf. Andersson and Sandler, op. cit.) that successful transplantation of fetal pancreatic tissue requires the explanted tissue to be kept in culture for some time before implantation. In addition to the tissue being kept in a viable state it is desirable to stimulate it for xcex2-cell growth and differentiation to promote excretion of insulin.
A method known in the art for preparation of fetal tissue particles (ICC, islet-like transplantation of pancreatic islets cell clusters containing xcex2-cells) comprises degradation of fetal pancreatic tissue with collagenase (cf. Andersson and Sandler, op. cit.). For various reasons comparatively low differentiated tissue is used for their preparation resulting in preparations containing few cells that immunochemically stain for insulin, and thus are identified as insulin-producing. On the other hand ICC have full potential for later differentiation at proper culture conditions. Such differentiation is considered, i.a., to be stimulated by human growth hormone, prolactin, and inhibitors of poly(ADP-ribose) synthetase, such as nicotine amide. In addition, the excretion of insulin itself can be stimulated. By nature D-glucose is the foremost stimulating agent but cell response in vitro to D-glucose can be low or nil, in particular with low-differentiated pancreatic tissue. Other agents, however, for instance inhibitors of phospho-diesterase, such as theophylline, stimulate the excretion of insulin at high glucose levels.
It is thus an object of the invention provide a method for the stimulation of insulin-producing xcex2-cells isolated from fetal or mature pancreatic tissue to produce and/or excrete insulin, the definition xe2x80x9cinsulin-producingxe2x80x9d cells also including cells competent for insulin production upon stimulation and/or differentiation, to keep such cells in a viable condition, to cultivate such cells, and to store and transport such cells. Another object of the invention is to provide a means for carrying out said method. Other objects of the invention are uses for such cells being stimulated, kept in a viable condition, transported or stored. A further object of the invention is an apparatus to be used in connection with cell stimulation, transport, storage or cultivation.
Further objects of the invention will become apparent from the study of the following summary of the invention, the description of preferred embodiments thereof, and of the appended claims.
In accordance with the present invention there is provided a method of the aforementioned kind comprising contacting insulin producing cells, in particular an aqueous suspension of insulin-producing cells, with a water-soluble cellulose derivative when being stimulated or stored, transported or cultured in preparation of stimulation. Such preparation of stimulation includes the preparation for transplantation. Preferred cellulose derivatives are alkylated, hydroxy-alkylated, and alkylated/hydroxyalkylated celluloses. Examples for such cellulose derivatives are hydroxyethyl cellulose, hydroxypropyl cellulose, methyl cellulose, ethyl cellulose, hydroxyethyl-methyl cellulose, hydroxyethyl-ethyl cellulose, hydroxypropyl-methyl cellulose. Most preferred is hydroxypropyl-methyl cellulose (HPMC). Aqueous solutions (true or colloidal) of the cellulose derivatives according to the invention are known to be viscous. The term cellulose derivative as used herein excludes derivatives of cellulose obtained by substantial degradation of the cellulose chain to fragments having a weight average molecular weight of 2,000 or less. The term cellulose derivative as used herein comprises pharmaceutically acceptable derivatives of cellulose obtained by substitution of hydrogen in cellulose hydroxyl groups by alkyl groups, preferably by C1-C5 alkyl groups.
Non-cellulose based carbohydrate polymers, such as alginates or agar (EP-A 363 125) or hyaluronic acid do not exert the substantial xcex2-cell stimulating effect according to the invention though providing aqueous solutions of substantial viscosity.
The aqueous solution of the cellulose derivative according to the invention can also contain other agents, such as nutrients in form of salts, amino acids, peptides, proteins, hormones or similar. Cell culture media of various kind can be provided with appropriate amounts of the cellulose derivative according to the invention whereby an aqueous xcex2-cell stimulating solution according to the invention is obtained.
In accordance with the invention are further disclosed uses for a xcex2-cell stimulating aqueous solution containing said cellulose derivative. These uses are preferably selected from culture, transport, transplantation, and storage of pancreatic cells.
In accordance with the invention there is also provided an apparatus for carrying out said method, the apparatus comprising a container for xcex2-cells containing an aqueous solution of the cellulose derivative according to the invention.
According to a second aspect of the present invention is disclosed a method for in-vitro cultivation of human adult and/or fetal cells, in particular cells considered difficult or impossible to cultivate in existing media, such as stem cells, neural cells, fibroblasts and skin cells, the method comprising contacting said cells being held in culture with a water-soluble cellulose derivative. Preferred cellulose derivatives are alkylated, hydroxy-alkylated, and alkylated/hydroxy-alkylated celluloses. Examples for such cellulose derivatives are hydroxyethyl cellulose, hydroxypropyl cellulose, methyl cellulose, ethyl cellulose, hydroxyethyl-methyl cellulose, hydroxyethyl-ethyl cellulose, hydroxypropyl-methyl cellulose. Most preferred is hydroxypropyl-methyl cellulose (HPMC).
According to a third aspect of the present invention is disclosed a method for the expression of peptides in bacteria, in particular E. coli, the method comprising contacting said bacteria with a water-soluble cellulose derivative. Preferred cellulose derivatives and examples thereof are given in the preceding paragraph.
According to a fourth aspect of the present invention is disclosed a method for wound healing, the method comprising contacting the wound with a water-soluble cellulose derivative. Preferred cellulose derivatives and examples thereof are given above.
According to a fifth aspect of the present invention is disclosed a method for dermal treatment aiming at the preservation of skin integrity and regeneration, the method comprising contacting the skin with a water-soluble cellulose derivative. Preferred cellulose derivatives and examples thereof are given above.
According to a sixth aspect of the present invention is disclosed a method for the expression of plasmids in bacteria, in particular E. coli, the method comprising contacting said bacteria when held in culture with a water-soluble cellulose derivative.
According to a seventh aspect of the present invention is disclosed a method for the preservation and/or transport and/or storage of tissue and organ transplants, the method comprising contacting the transplant with a preservation and/or transport and/or storage effective amount of a water soluble cellulose derivative.
According to an eight aspect of the present invention is disclosed a method for promoting the healing of the respiratory tract epithelium including that of the larynx, trachea, bronchi and pulmonary epithelium, the method comprising administration of a therapeutically effective amount of a water-soluble cellulose derivative in form of microdroplets of its aqueous solution provided, for instance, by a nebulizer.
Contacting with a water soluble cellulose derivative includes contacting with an aqueous solution of the derivative.
According to a ninth aspect of the invention is disclosed a method for in-vitro stimulation of eucaryotic cells being held in culture, said stimulation comprising accelerating cell maturation or excretion of metabolic products, the method comprising contacting said cells with a cell maturing or cell stimulating amount of a water-soluble cellulose derivative.
According to a tenth aspect of the invention is disclosed a method for in-vitro cultivation of eucaryotic cells being held in culture, the method comprising contacting said cells with a water-soluble cellulose derivative.
The means according to the invention for carrying out the aforementioned methods comprises a water soluble cellulose derivative. Preferred water soluble cellulose derivatives are alkylated, hydroxy-alkylated, and alkylated/hydroxy-alkylated celluloses. Examples for such cellulose derivatives are hydroxyethyl cellulose, hydroxypropyl cellulose, methyl cellulose, ethyl cellulose, hydroxyethyl-methyl cellulose, hydroxyethyl-ethyl cellulose, hydroxypropyl-methyl cellulose. Most preferred is hydroxypropyl-methyl cellulose (HPMC).
While not being bound by any hypothesis in regard of the beneficial effect of the water-soluble cellulose derivative of the invention, this effect on living cells, cell aggregates, tissues, and organs for transplantation is thought to be due to the aqueous solution of the cellulose derivative providing an environment which is more naturalxe2x80x94more structuredxe2x80x94than the aqueous environment provided by known culture media.
Further advantageous and preferred features of the invention are disclosed in the appended claims and the following description of preferred embodiments of the invention, said non-limitative embodiments however only being provided for illustrative purposes.